Research ArticleOsteoarthritis

Proteoglycan 4 Expression Protects Against the Development of Osteoarthritis

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Science Translational Medicine  13 Mar 2013:
Vol. 5, Issue 176, pp. 176ra34
DOI: 10.1126/scitranslmed.3005409
  • Fig. 1

    Prg4 transgenic mice are protected from development of age-related OA. (A) Comparison of 10-month-old WT mice and Prg4 transgenic mice knee joints by OARSI grade (*P < 0.05, Wilcoxon rank test; n = 7). (B) Safranin O staining and immunohistochemistry (antibody used to the left) of 10-month-old WT and Prg4 transgenic mice. Black arrows indicate OA changes in proximal tibial articular cartilage in sagittal section. Scale bars, 100 μm. (C) Representative image of the reconstruction of articular cartilage in 10-month-old WT and Prg4 transgenic mouse with femoral cartilage shown in blue and tibial cartilage shown in yellow. Red arrow indicates loss of cartilage. Scale bar, 500 μm. (D) Quantification of articular cartilage volume and surface area of bone covered by cartilage in mouse knee joints by phase-contrast μCT (*P < 0.01, t test; n = 5). Error bars indicate SEM.

  • Fig. 2

    Prg4 transgenic mice are protected from the development of posttraumatic OA. (A) Comparison of Prg4 transgenic mice and WT mice knee joints by OARSI grade [*P < 0.05, analysis of variance (ANOVA); n = 8]. (B) Safranin O staining and immunohistochemistry (antibody used listed above each column) of WT sham (WT Sham), WT with transection (Wt Sx), and Prg4 transgenic mice with transection (Prg4 Sx). Black arrows indicate areas with OA changes in saggital sections through the knee. Scale bars, 100 μm. (C) Representative images of reconstruction of knee joints with phase-contrast μCT with femoral cartilage shown in blue and tibial cartilage shown in yellow. Red arrow indicates loss of cartilage. Scale bar, 500 μm. (D) Quantification of articular cartilage volume and surface area of bone covered by cartilage in mouse joints by phase-contrast μCT (*P < 0.01, **P < 0.05, ANOVA; N.S., not significant; n = 5 to 6). (E) Average time that mice stayed on rotating rod 2 months after CLT in rotarod analysis (*P < 0.05, ANOVA; n = 15). (F) Response time of mice after placement onto a 55°C platform in hotplate analysis (*P < 0.05, ANOVA; n = 10). Error bars indicate SEM.

  • Fig. 3

    Prg4 delivered by HDVs protects mice from development of OA. (A) Representative image comparing intra-articular injection of HDV and different serotypes of AAV. Different vectors (109 vp) each expressing GFP were injected intra-articularly into the knee joint. The lower panels are enlarged images of the boxed areas in the upper panels. Green, GFP; blue, 4′,6-diamidino-2-phenylindole; C, cartilage; S, synovium. Scale bars, 100 μm. (B) Comparison of luciferase expression after intra-articular injection of FGV and HDV (n = 6) into the knee joint. (C) Representative image of expression patterns of intra-articular injections of different doses of HDV encoding β-galactosidase. The bottom images are enlarged areas of the black box in the upper images of saggital sections through the knee joint. Scale bars, 100 μm. (D to G) Scheme of experiment comparing the preventive effect of HDV-PRG4 injection and evaluation by OARSI grade and cartilage volume (D). Before transection, WT mice were injected with HDV-PRG4 intra-articularly at 109 vp per joint (Sx + PRG4 H) or 108 vp per joint (Sx + PRG4 L). Sham, transection without treatment (Sx), and injection of virus without transgene before transection (Sx + Vector) served as controls. Degree of OA is presented by OARSI grade (E), cartilage volume (F), and cartilage surface area (G) (*P < 0.05, ANOVA; n = 8 to 10 in OARSI grading; n = 5 to 6 in cartilage volume analysis). (H to K) Scheme of experiment comparing the protective effect of HDV-PRG4 injection and evaluation by OARSI grade and cartilage volume (H). Two weeks after transection, WT mice were injected with HDV-PRG4 intra-articularly at 109 vp per joint (Sx + PRG4 H) or 108 vp per joint (Sx + PRG4 L). Sham, no treatment (Sx), and HDV-GFP injection (Sx + GFP) served as controls. Degree of OA is presented by OARSI grade (I), cartilage volume (J), and cartilage surface area (K) (*P < 0.05, ANOVA; n = 8 to 10 in OARSI grading; n = 5 to 6 in cartilage volume analysis). Error bars indicate SEM.

  • Fig. 4

    Prg4 delays OA by inhibiting cartilage catabolism and terminal hypertrophy. (A) Microarray heat map analysis comparing superficial zone cartilage of WT and Prg4 transgenic mice. Genes with expression changes larger than 1.5-fold and P value less than 0.05 are plotted. (B) Transcription factor activity changes predicted by Ingenuity Pathway Analysis. All transcription factors shown here were predicted to be suppressed by Ingenuity Pathway Analysis. X axis indicates the number of genes/gene groups controlled by each transcription pathway in the gene list submitted. (C) Changes in gene expression (Prg4, Hif3a, Vegf, Col10a1, and Mmp13) in C3H10T1/2 cells under hypoxia (1% oxygen for 8 hours). Cells are sham-treated and infected with empty HDV (vector) or with HDV-PRG4 (PRG4) (*P < 0.05, ANOVA; n = 3). (D) Changes of Prg4 and Hif3α expression under normoxia and hypoxia in TC71 Ewing sarcoma cells (*P < 0.05, t test; n = 3). (E) Changes of gene expression after Hif3α knockdown by small interfering RNA (siRNA) (*P < 0.05, ANOVA; n = 3). (F) Proposed model of PRG4 function in prevention of OA development. Error bars indicate SEM.

Additional Files

  • Supplementary Material for:

    Proteoglycan 4 Expression Protects Against the Development of Osteoarthritis

    Merry Z. C. Ruan, Ayelet Erez, Kilian Guse, Brian Dawson, Terry Bertin, Yuqing Chen, Ming-Ming Jiang, Jason Yustein, Francis Gannon, Brendan H. L. Lee*

    *Corresponding author. E-mail: blee@bcm.tmc.edu

    Published 13 March 2013, Sci. Transl. Med. 5, 176ra34 (2013)
    DOI: 10.1126/scitranslmed.3005409

    This PDF file includes:

    • Fig. S1. Prg4 overexpression under the Col2a1 promoter does not adversely affect the development of mice.
    • Fig. S2. Generation and characterization of HDV-PRG4.
    • Fig. S3. Gene expression profiling of wild-type versus Prg4 transgenic superficial layer chondrocytes.
    • Data set 1. List of genes showing more than 1.5-fold change in the microarray analysis.
    • Data set 2. List of transcription factors predicted by Ingenuity Pathway Analysis to be activated or inhibited.

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